Pharmaceutical, biotechnology, or genomics companies use DNA analysis systems for target identification and drug screening in pharmaceutical drug discovery. In many of these systems, biomolecules (e.g. DNA, RNA, cDNA, Proteins) labeled with various dyes hybridize to chips that offer different molecular counterparts for hybridization of e.g. single stranded RNA in different areas of the chip. A scanner is then used to read the fluorescence of these molecules under illumination with suitable (most often laser) light. The scanner acts like a large field fluorescence microscope in which the fluorescent pattern caused by hybridization of labeled molecules is scanned on the chip. In particular, a laser induced fluorescence scanner provides for analyzing large numbers of genes/mutations/alleles in a biological sample. For various reasons it is often desirable to have samples labeled with different dyes hybridize (competitively) to the same chip or "sample carrier". In this case, a scanner needs to be able to be able to differentiate between the different kinds of molecules with as little crosstalk as possible.
U.S. Pat. No. 5,091,652 entitled "Laser Excited Confocal Microscope Fluorescence Scanner and Method" teaches a scanner for sequentially scanning the fluorescence from a series of labeled samples on a sample carrier with a confocal microscope. A single laser is employed for illuminating a single volume of a gel sample carrier and for receiving and processing fluorescence emissions from the volume to provide a display of the separated sample.
The Hewlett-Packard G2500A is a fluorescence scanner that employs a single laser with two filters for sequential multiple frequency scanning of fluorescently labeled chips in which two dyes may be applied to the sample. Crosstalk between the emission spectra from the two dyes reduces the signal to noise ratio of any detected signal. In particular. if the signal from one dye is very strong and the signal from the other dye is very weak, crosstalk between the two channels may severely limit the performance of a given system. While crosstalk can be reduced in some instances by first scanning with one laser and then illuminating the sample with another laser, the sequential registration of two full scans increases scan time to almost twice that of existing systems.
U.S. Pat. No. 5,294,799 discloses a microfluorometer which simultaneously excites one or more fluorophores with two or more wavelengths. The intensity of the excitation at each wavelength is time modulated at a separate frequency and a separate frequency-locked phase sensitive detector for each modulation frequency allows discrimination of the contribution from the individual spectra corresponding to each fluorophore. However. amplitude modulation at its best (i.e. for 100% contrast), results in a 50% reduction of the average laser power incident on the spot to be measured. This degrades signal performance either by increased shot noise or by increased saturation of the dye molecules at the peak of the modulation cycle. It may also result in the need for a more powerful laser source which is likely to increase cost.
The Scanarray 3000 scanner manufactured by the General Scanning Corporation and the Avalanche scanner manufactured by the Molecular Dynamics company each employ two lasers that are used to sequentially scan a chip that results in rather long scan times limiting throughput in many applications.
There exists a need for a laser induced scanner that can quickly determine the ratio(s) of the signals caused by two or more dyes. A need also exists to do such an analysis over a very wide dynamic range of that ratio (e.g. to a range up to going from 1:10000 to 10000:1) with a defined minimum signal-to-noise ratio of the ratio measurement.
It would be desirable and of considerable advantage to provide a multiple frequency scanner that differs from those employed in the prior art by reducing both crosstalk and scan time.